Rhodotorula toruloides for carotenoid production using waste hardwood biomass

Rhodotorula toruloides for carotenoid production using waste hardwood biomass | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rhodotorula toruloides for carotenoid production using waste hardwood biomass Stefano Bertacchi, Francesca Sabatini, Giovanni Maria Bernardini, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8187172/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Mar, 2026 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted 11 You are reading this latest preprint version Abstract This work explores the potential of underutilized urban pruning residues from hardwood as feedstocks for bioprocesses based on the carotenogenic yeast Rhodotorula toruloides , investigating the correlation between biomass composition and carotenoids production. Enzymatic hydrolysates from woods and barks of sessile oak and mulberry tree were used as substrates for microbial fermentation, obtaining superior titers and productivity of β-carotene and torulene when compared to previous published processes. Mulberry tree bark hydrolysate yielded the highest total sugars (16.4 g/L), but sessile oak bark hydrolysate showed the highest β-carotene production (362.7 mg/L) after 30 hours of fermentation. Woody biomasses are known to contain significant amounts of extractive inhibitory compounds. Surprisingly, when we removed them in order to promote growth and production, we observed a significant drop in carotenoids titers, which resulted in line with published productions from biomasses lacking extractive components. These data suggest that stressful compounds present in the extractive fractions are crucial for promoting high production and productivity, when compared with the use of biomasses lacking such components. This research therefore highlights the potential of underexplored urban woody residues, thanks to the presence of triggering components, as an advantageous feedstock for microbial carotenoids production. Rhodotorula toruloides Carotenoids Biodiversity valorisation Biorefinery Lignocellulosic biomass Enzymatic hydrolysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The exploration of diverse biological feedstocks and microorganisms is crucial for establishing efficient and environmentally friendly biorefinery and bioconversion pathways 1 , 2 . Woody biomasses, derived from pruning and forestry, agricultural wastes, as well as dedicated energy crops, constitute a largely untapped resource for biorefineries due to their prevalent lignocellulosic composition. These renewable materials offer in form of their comprised polymers cellulose, hemicelluloses, and lignins valuable alternatives to fossil resources for the production of fuels, chemicals, and high-value bioproducts within the bioeconomy scenario 3 – 5 . The integration of microbial biotechnology into biorefinery processes can be a key factor to unlock the full valorisation potential of woody biomasses. Enzymatic hydrolysis enables the release of fermentable pentose and hexose sugars from the cellulose and hemicellulose fractions, providing carbon sources for microbial growth and biotransformation. Among the diverse microbial candidates for lignocellulosic biomass valorisation, the oleaginous yeast Rhodotorula ( Rhodosporidium ) toruloides has gained considerable attention. This yeast exhibits a remarkable ability to metabolize a wide range of sugars derived from lignocellulosic hydrolysates, i.e. , both pentose and hexose sugars, and has demonstrated tolerance to various inhibitory compounds that can arise during biomass pretreatment 6 – 10 . Furthermore, R. toruloides is a lipidogenic yeast and natural producer of valuable carotenoid pigments, including β-carotene and torulene. This class of compounds features significant antioxidant and provitamin A activity, and colouring properties with applications in food, feed, cosmetic, and pharmaceutical industries 7 , 8 , 11 , 12 . The global market for carotenoids continues to grow (USD 1.48 billion in 2023, projected to grow at a CAGR of 3.5% to 2030), driven by increasing consumer demand for natural and health-promoting ingredients, as well as bio-based colorants, with ꞵ-carotene products accounting for a 24.7% revenue share in 2023 13 . The torulene market, although smaller, is attracting an increasing interest due to its biological properties and potential health benefits 14 Despite the growing insight into the capabilities of R. toruloides in the context of lignocellulosic biomass valorisation, research that specifically investigates the utilization of novel woody feedstocks from local biodiversity for carotenoid production is yet underexplored. While several studies have examined R. toruloides growth on conventional agricultural residues (Table 1 ), the potential of underutilized woody forestry residues deserves further investigation. Recent studies in the literature deploying woody hydrolysate as possible substrates for R. toruloides , are either centred on lipid rather than carotenoid production, or they report the introduction of additional carbon and/or nitrogen sources 15 – 17 . Additionally, most of the recent publications on the topic are focused on the valorisation of by-products from pruning, mainly of olive trees 18 – 20 or vines 21 – 23 , whereas urban pruning residues are exploited mainly for energy or chemical production via physical and/or chemical treatments 24 – 26 . Thus, there is a lack of studies on the valorisation of such urban residues by microbial action, beyond composting or anaerobic digestion 4 , 24 , 25 Therefore, since the current use of such residues for low value products ( i.e. energy), this study aims to providing alternative valorisation for these lignocellulosic biomasses of urban origin to obtain a product with an higher value ( i.e. carotenoids). Table 1 Examples from literature for the production of carotenoids from residual biomasses by the use of R. toruloides strains, with a focus on the comparison between productivities obtained. SOW , SOB , MTW , and MTB , as disclosed in the text, represent wood ( W ) and bark ( B ) of sessile oak ( SO ) and mulberry tree ( MT ). R. toruloides strain Biomass Treatment Added N source Starting sugars (g/L) Time (h) Carotenoid Production (mg/L) Carotenoid productivity (mg/L/h) Reference DSM 4444 Defatted waste wheat bran Biological (Cellic® CTec 3) Yeast extract, ammonium sulfate 46 144 180 1.24 27 ACCC20341 Tea waste Chemical (Sulfuric acid) Yeast extract, ammonium sulfate 95.63 96 180 1.875 28 C23 Synthetic lignocellulosic-like medium - Yeast extract, ammonium sulfate 120 144 11 0.07 29 L/24-26-1 Sugarcane molasse Chemical (Sulfuric acid) Yeast extract, ammonium sulfate 40 168 21.5 0.128 30 NCIM 3547 Neem oilseed cake Chemical (Microwave mild acid hydrolysis) Yeast extract 25.83 96 60.88 0.88 31 DSM 4444 Spent coffee grounds Biological (β-glucosidase Viscozyme) Yeast extract 23.3 125 13 0.104 32 DSM 4444 MTW MTB SOW SOB eMTB eSOB Biological (Novozymes NS22119) None 9.1 15.7 7.7 13.8 13.3 12.1 30 89.9 163.5 123.8 362.7 10.6 25.27 2.9 5.5 4.1 12.1 0.35 0.84 This study Table 1 Examples from literature for the production of carotenoids from residual biomasses by the use of R. toruloides strains, with a focus on the comparison between productivities obtained. SOW , SOB , MTW , and MTB , as disclosed in the text, represent wood ( W ) and bark ( B ) of sessile oak ( SO ) and mulberry tree ( MT ). eMTB and eSOB represent the corresponding biomasses without the extractive components. This study aims consequently at addressing the potential of urban pruning/forestry residues in the form of sessile oak ( Quercus petraea , SO ) and mulberry tree ( Morus alba , MT ) biomasses, sourced from an urban park within the Milan metropolitan area, as substrates for R. toruloides growth and carotenoid production. The initial steps of this work involve the hydrolysis of the residual biomasses to obtain growth media for the yeast, followed by the analysis of the resulting sugar and nitrogen concentrations from both wood ( W ) and bark ( B ) of sessile oak and mulberry tree, i.e. , SOW , SOB , MTW , and MTB . Furthermore, the woody hydrolysates were presented to R. toruloides in a separated hydrolysis and fermentation ( SHF ) setup. Differences in carotenoid production during fermentation time were analysed, with particular attention to identify components of the woody biomass that might trigger or inhibit carotenoid biosynthesis. 2. Materials and methods 2.1. General information All chemicals used were analytical grade. Reagent grade water was provided by a Milli-Q® Integral 5 purification system (Merck KGaA, Darmstadt, Germany). Dichloromethane, toluene, ethanol, ethyl acetate and acetone (GC-MS grade) were purchased from Carlo Erba (Cornaredo, Italy). Sulphuric acid (95–97%) was obtained from Merck KGaA (Darmstadt, Germany). Acetonitrile (LC-MS grade) was supplied from VWR International SrL (Milan, Italy). Pure standards of β-carotene and caffeine were obtained from Sigma-Aldrich (St Louis, MO, USA), while the standard of torulene was provided by CaroteNature (Münsingen, Switzerland). 2.2. Biomass collection and preparation The residual woody biomasses used in this study are sessile oak ( Quercus petraea ) wood ( SOW ) and bark ( SOB ), as well as mulberry tree ( Morus alba ) wood ( MTW ) and bark ( MTB ). These materials were collected as leftovers from the maintenance of Parco della Besozza (Pioltello, Lombardy), an urban park in the metropolitan area of Milan (Italy). These biomasses were used as obtained, e.g. , in the form a conventional urban pruning/forestry workflow that produced them in an only roughly separated form between bark and wood. Consequently, both starting materials mutually contain residues of the other component, i.e. , bark or wood. Importantly, these biomasses were applied in this study without any upstream detoxification step, yielding the possibility to explore eventual inhibiting factors. The biomasses were dried and stored at room temperature till processing. Woody biomasses were mechanically grounded to a final particle size of less than 1 mm. The resulting powdered biomass was used for all the steps of treatment and analysis. 2.3. Scanning electron microscope (SEM) analysis of grounded biomasses SEM-EDS observations were performed at the Platform of Microscopy of the University of Milano-Bicocca (PMiB) with a field emission gun (FEG) SEM Zeiss Gemini 500, operating at 3 and 5 keV and equipped with a Bruker QUANTAX integrated wave-dispersive/energy-dispersive (WDS/EDS) system. Metallization was carried out using an Edwards Sputter Coater S150B, with Au/Pd target (70% Au/30%Pd) in Ar atmosphere, current of 40 mA, voltage 1 kV, suttering time 1 min, and depositing a 10 nm thick Au/Pd layer. 2.4. Removal of extractives from grounded biomasses Wood or bark powder was placed into a crucible glass filter, pore size III, for subsequent solvent extraction using a standard 100 mL Soxhlet extractor. The first extraction utilized 150 mL of dichloromethane (DCM) for 6 h, followed by a second extraction using 150 mL of a 1:1 (v/v) toluene/ethanol (tol/EtOH) mixture for 12 h. The final extraction used 150 mL of acetone for 6 h. The extracts were subsequently isolated by removing the solvents in vacuo . 2.5. Determination of acid-insoluble and acid-soluble lignin content through Klason extraction For the determination of acid-insoluble lignin, a NREL procedure was adopted (Lin and Dence 1992). Approximately 100 mg of milled wood or bark was carefully weighed into a test tube. To this, 2 mL of 72% (w/v) sulfuric acid (H 2 SO 4 ) solution was added at room temperature, with intermittent stirring over a 2 h. The resulting solution was then diluted with deionized water to achieve a 3% (w/v) H 2 SO 4 concentration. Then, the solution was transferred to a sealed flask and subjected to heating at 120°C and 2 bar for 7 min using an autoclave (LaborAutoklav, Certoclav, Austria). The solution was filtered through a crucible of size M, washed with deionized water until a neutral pH was attained, and the crucibles were placed in an oven at 105°C for 12 h to fully dry the sample. The determination of acid-insoluble lignin was performed gravimetrically. For the assessment of acid-soluble lignin, the filtrate obtained from the acid-insoluble lignin procedure was diluted to 100 mL using deionized water. The quantification of acid-soluble lignin was performed by calculating UV absorbance at 205 nm, utilizing an extinction factor of 113 L∙g − 1 ∙cm − 1 , which was chosen according to literature for hardwoods (Lin and Dence 1992). 2.6. Enzymatic hydrolysis of woody biomasses. Enzymatic hydrolysis of the woody biomasses was performed using the enzyme cocktail NS22119, kindly provided by Novonesis (Novonesis A/S, Copenhagen, Denmark). NS22119 contains a wide range of carbohydrases, including cellulase, arabinase, pectinase, β-glucanase, hemicellulase, and xylanase from Aspergillus aculeatus , as described by the producer. As a first step, 15% of biomass (w/v) was autoclaved at 121°C for 1 h to both sterilize and mildly pre-treat the biomass. Afterward, NS22119 enzyme mix (11.9% w/w biomass ) was added to the biomass and incubated at 50°C in a water bath under mild agitation (105 rpm) for 6 h. At the end of the process, the hydrolysates were centrifuged (8000 rpm, 10 min) to precipitate the insoluble components and collect the supernatant to be used as growth medium for R. toruloides . 2.7. Microbial strain, media and growth conditions Rhodotorula (Rhodosporidium) toruloides DSM 4444 was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, GmbH) and kept in cryotubes at − 80°C in 20% glycerol (v/v). The composition of the medium (YPD) for the pre-inoculum was as follows: 20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose. Yeast extract and peptone was purchased from Biolife Italia S.r.l. (Milan, Italy). All other reagents were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). After plating on YPD, a pre-inoculum was run in rich medium until the stationary phase. Then, cells were inoculated at 0.25 OD in shake flasks at 30°C and 160 rpm in SHF setting using woody hydrolysates. Growth was measured in terms of optical density (OD) at 600 nm. Samples (1 mL) were collected during the growth and centrifuged (8000 rpm, 5 min). The supernatant was used for sugar quantification, whereas the cellular pellet was used for carotenoid extraction. 2.8. Intracellular carotenoids extraction Acetone was used for extracting carotenoids from R. toruloides cells as described elsewhere (Bertacchi et al. 2020). In brief, 1 mL of cells was collected and harvested by centrifugation at 10000 rpm for 7 min, and the pellet was then resuspended in 1 mL acetone and broken using glass beads by thorough agitation with a FastPrep-24™ (MP Biomedicals, LLC, Santa Ana, CA, USA) with 3 cycles of 30 sec each at 4°C. Carotenoids were thus extracted in the acetone phase, collected in the supernatant by centrifugation and stored at -20°C. The extraction was repeated with a new aliquot of acetone until the biomass turned colourless. 2.9. Characterization of woody hydrolysate using dinitrosalicylic (DNS) test The dinitrosalicylic (DNS) test was used to determine the total reducing sugar content of the obtained hydrolysed. An optimized and miniaturized DNS protocol was deployed. 35 DNS reagent consists of: 10 g/L 3,5-dinitrosalicylic acid (Sigma-Aldrich), 16 g/L NaOH (Merck), 30 g/L potassium sodium tartrate (Carlo Erba). For quantification, a glucose calibration curve (30–0.6 g/L) was prepared. In each tube, 190 µL of the DNS reagent and 10 µL of sample were inserted and then vortexed. The tube was heated at 100°C for 2 min and then cooled to room temperature. Following heat treatment, all samples were transferred in 96-well plates and analysed at 531 nm in a multiplate reader (VICTOR X3, PerkinElmer). Specific sugars (glucose, fructose, sucrose, xylose, galactose) were quantified by using the spectrophotometric enzymatic kits K-SUFRG, K-XYLOSE and K-ARGA (Megazyme, Southern Cross Rd, Bray, Co. Wicklow, Ireland), following the manufacturer’s instructions. For this purpose, samples were treated by the addition of 20 g/L of polyvinylpolypyrrolidone (PVPP), shaken vigorously for 5 min and then filtered. Nitrogen content in the form of primary amines was quantified by the use of PANOPA enzymatic kit (Megazyme). 2.10. Determination of yields and C/N ratio “Yield of carotenoids on initial biomass” was calculated as the ratio between the carotenoids production and the amount of biomass used in the enzymatic hydrolysis. Similarly, “yield on hydrolysate sugars” or “yield on consumed sugars” have as denominator the amount of sugars in the hydrolysate provided to cells as growth medium or the amount of sugars effectively consumed after 30 h of growth, respectively. C/N ratio of hydrolysate was calculated as the ratio between the carbon content in the sugars measured by DNS assay and the measured primary nitrogen source. The carbon content was calculated by multiplying total reducing sugars amount by 0.4, as 40% of a glucose and fructose molecule is carbon (and most of the sugars in the media are either glucose or fructose). 2.11. Characterization of woody hydrolysate using 1 H nuclear magnetic resonance (NMR) spectroscopy To a 500 µL aliquot of the hydrolysate solution were added 100 µL D 2 O as lock-triggering solvent. A Bruker 400 MHz spectrometer controlled via TopSpin 4.1.4 with a 5 mm double resonance broadband inverse probe was used to acquire 1 H NMR spectra at 30°C. The Bruker zgesgp pulse sequence was used with NS = 48, applying the presaturation method for water signal suppression. MestreNova Version 9.0.1 (Mestrelab Research S.L.) was used for data processing. 2.12. Characterization of woody hydrolysate using 1 H- 13 C heteronuclear single quantum coherence (HSQC) analysis To a 500 µL aliquot of the hydrolysate solution 100 µL D 2 O were added as lock-triggering solvent. A Bruker 400 MHz spectrometer controlled via TopSpin 4.1.4 with a 5 mm double resonance broadband inverse probe was used to acquire HSQC spectra at 30°C. The Bruker hsqcetg pulse program in the DQD acquisition mode was applied with NS = 64; TD = 2048 (F2) and 512 (F1); SQ = 12.9869 ppm (F2) and 164.9996 ppm (F1); O2 (F2) = 2601.36 Hz and O1 (F1) = 7799.05 Hz; D1 = 2 s; CNST2 1 J(C-H) = 145; and acquisition time F2 channel = 197.0176 ms and F1 channel = 15.4164 ms. For each sample, the pulse length of the 90° P1 high-power pulse was optimized. MestreNova Version 9.0.1 (Mestrelab Research S.L.) was used for data processing. 2.13. Characterization of wood and bark extracts using gas chromatography coupled with mass spectrometry (GC/MS) Gas Chromatographic-mass spectrometric analyses of the dried extractives were performed by re-dissolving a dried sample of the extracts in 500 µL of dichloromethane. The chromatographic separations were performed using a Shimadzu GCMS QP2020NX (Shimadzu Corporation, Kyoto, Japan) equipped with Shimadzu autosampler AOC20i. An SH-Rxi-5ms fused silica capillary column (stationary phase (5%-Phenyl)-methylpolysiloxane, 30 m x 0.25 mm i.d., 0.25 µm, Shimadzu Corporation, Kyoto, Japan) was used as stationary phase, and He (UHP grade) as carrier gas. The system was operated in ‘linear velocity mode’ with a starting pressure of 100 kPa, 280°C injection temperature, and 280°C interface temperature. The injection volume was 2 µL, the injection port operated in splitless mode. The temperature program was set as follows: the initial temperature of 50°C was held for 1 min, then increased at a rate of 10°C min ˗ 1 to a 280°C, which was maintained for 15 min. The MS operated in electron ionization mode (EI) at 70 eV, acquiring in full-scan mode in the m/z range of 50–500. LabSolutions–GCMS Version 4.54 software (Shimadzu Corporation) was used as system control, instrument management and data acquisition. Substances were identified using NIST MS Search, version 2.4 (2020). 2.14. Carotenoids quantification using flow injection analysis coupled by mass spectrometry (FIA/MS) Following acetone-based extraction of carotenoids described in Section 2.8 of the Materials and Methods, quantitative analysis of β-carotene and torulene was performed on the resulting extracts. β-carotene and torulene were used as analytical standard and caffeine as internal standard (10 ppm) to construct five-point calibration curves (concentration range 0.01-1 ppm for torulene, 0.5–20 ppm for β-carotene). The calibration curves were obtained in flow injection analysis (FIA) mode, which was applied even for the analysis of the extracts in acetone, previously centrifuged at 1500 rpm for 5 min. A binary LC 20AT pump, coupled with an SPD M20A UV system and a 2010 EV single-quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source was used. All modules are Shimadzu Corporation (Kyoto, Japan). The mobile phase was set at 100% acetonitrile, with a flow rate of 0.3 mL/min, and an injection volume of 20 µL. The operating conditions of the ESI-MS system were as follows: nebulizer gas (N 2 , purity > 98%) with flow rate of 1.5 mL/min, curtain desolvation line (CDL) at 240°C and heat block (HB) at 230°C, capillary voltage 2.5 kV. Acquisition of MS spectra was performed in positive ion mode in selected ion monitoring (SIM), observing fragments at m/z 195 for caffeine, 534 for torulene, and 536 for β-carotene. LCMS solution Version 3.30 268 software (2002–2005; Shimadzu Corporation) was used for instrument management and data acquisition. 3. Results and discussion 3.1. Characterization of woody biomass The complex nature of residual woody biomass is a well-known initial hurdle in defining its application in a microbial-based biorefinery 1 , 36 , 37 . Additionally, the intrinsic biodiversity, in terms of plant species and parts, is key to highlight differences in carotenoids production possible arising from the procedure steps as hydrolysis and fermentation ( SHF ). For these reasons, the composition of the biomass selected for this study, i.e. , sessile oak wood ( SOW ) and bark ( SOB ), as well as mulberry tree wood ( MTW ) and bark ( MTB ) was initially characterized. Table 2 summarises the main components constituting these lignocellulosic biomasses, in terms of the sugar-fraction vs. the lignin content, and considering the extractable compounds from the three solvent consecutive extractions. Table 2 Composition of biomasses used in this study. Cellulose & hemicellulose [% (w/w)] are calculated as the difference of the sum of lignin (acid soluble), lignin (acid insoluble) and total extractives. Total extractives [% (w/w)] is given by the sum of DCM, EtOH/tol and acetone consecutive extractions. Biomass component SOW SOB MTW MTB Cellulose & hemicellulose [% (w/w)] 61 ± 11 55 ± 5 59 ± 9 53 ± 5 Lignin (acid soluble) [% (w/w)] 4 ± 1 3.7 ± 0.8 1.5 ± 0.1 2.8 ± 0.6 Lignin (acid insoluble) [% (w/w)] 28 ± 3 31 ± 2 32 ± 3 35 ± 2 Total extractives [% (w/w)] 6.4 ± 0.2 10.5 ± 0.3 7.4 ± 0.4 8.9 ± 0.8 Overall, the delineated compositions are in line with what is expected from hardwoods, with less cellulose contents in the barks 38 Bulk sugar contents are rather similar, both between the wood components and the bark ones. The analysis of the lignin content of the various biomasses (Table 2 ) shows values that seem high for hardwood species. It can be assumed, based on literature data (Kögel-Knabner 2021), that a low percentage of the determined insoluble Klason lignin content is actually comprised of humins, known to form upon sugar degradation during Klason analysis and co-precipitating with the insoluble lignin. Interesting is the fact that in the barks is found more lignin than in the bulk. This must be explained by the residual wood in the bulk bark material and the presence of suberins that behave eventually similar to the lignin in the applied NREL procedure 40 Detailed analysis of the various extractives obtained was performed using gas-chromatography coupled to mass spectrometry to provide an in-depth characterization of their composition. Indeed, rather scant information is reported in the literature on the composition of the trees, wood and bark under study. The results, detailed in Table S1 , show the expectable range of bioactive small molecules, with a slightly richer composition found for two bark samples, i.e. , SOB and MTB . Several molecular classes of compounds have been identified in the extracts, mostly fatty acids and sterols, but also marker species belonging to other chemical classes. Mulberry tree resulted to be richer in extractives than sessile oak, showing high amount and variety of fatty acids, phenolic compounds, vitamins and coumarins especially in the bark. The three isomers of tocopherol (δ-tocopherol, γ-tocopherol, α-tocopherol), an antioxidant compound known as vitamin E, has been already observed in mulberry fruit and bark 41 , as well as coumarins especially in leaves 42 but not those detected in our study as umbelliferone, methyl ostruthin, ostruthin and esculetin typical of umbelliferae family of plants. As regards sessile oak, SOW profile is particularly poor while SOB interestingly presents the wider variety of terpenoids of all the four samples. The identified terpenes belong to the family of diterpenes (copalol), tritepenes (β-amyrone, 24-norursa-3,12-diene, lupeol, β-amyrin, glutinol, copalol and simiarenol) and sesquiterpenes (Humulenol-II). SOB extractives show also the unique presence of alcohols, alkenes and other compounds such as benzaldehyde and benzaldehyde diethylacetal, compared to the other biomasses. 3.2. Enzymatic hydrolysis of woody biomasses To unlock the potential of lignocellulosic biomasses in microbial-based biorefineries, an enzymatic cocktail able to release both hexose and pentose into the liquid phase sugars is commonly used. As described in Section 2.6, powdered biomasses, i.e. , SOW , SOB , MTW , and MTB , were subjected to enzymatic hydrolysis. Data reported in Fig. 1 A clearly show the effect of the action of such an enzymatic cocktail in terms of total reducing sugars, with an increase in the carbohydrates released from the sole pre-treatment. Specifically, given the same initial amount of biomass processed per volume (15% w/v), data show that MTB is the raw material providing the highest amount of released sugars (16.4 ± 0.9 g/L), whereas on MTW basis the lowest content is obtained, reaching approx. only 50% of that seen for the MTB sample (8.3 ± 0.5 g/L). Interestingly, the bark moiety provided also for the SO -biomass consistently more sugars compared to the corresponding wood, i.e. , SOW . This fact can be related to the different structural characteristics of bark with respect to wood. Scanning electron microscopy (SEM) imagines collected on the various biomasses after grounding show that the bark samples seem slightly more porous, exposing thus a larger surface for the enzymes to work on (Fig. 2 ). Nevertheless, these data are in contrast with previous research suggesting a negative impact of bark presence towards enzymatic saccharification, using as biomass source residues of spruce, birch, and douglas-fir 43 , 44 In order to investigate the composition of the carbohydrate mixture in the hydrolysates of the various biomasses, 1 H- 13 C HSQC analyses were performed alongside quantification by wet-chemical assessment by the use of enzymatic assays of most of the sugars identified. Tables 4 and 5 report the results obtained in the assays, whereas Fig. 3 shows the HSQC spectra obtained for the various hydrolysates. Table 4 Carbohydrates and inhibitors identified in hydrolysates after 6 hours of the biomasses used in this study by means of 1 H- 13 C HSQC analyses ( HSQC ) and. A full list of identified signals and their respective shifts is given in the Supporting Information. Carbohydrate colour code SOW SOB MTW MTB Arabinose --- --- traces traces Fructose X X X X Galactose X X X X Glucose X X X X Mannose X X X X Methyl gluconoric acid traces traces traces traces Xylose traces traces X X Other compounds Alkanoyl residues X X X X Alkyl residues traces traces traces traces Gallate residues X X traces traces Pyrogalloyl residues X X X X Table 5 Carbohydrates identified in hydrolysates after 6 hours of the biomasses used in this study by means of tested enzymatic assays. Values are the means ± SD of three independent experiments. Titer (g/L) SOW SOB MTW MTB Glucose 2.4 ± 0.1 1.5 ± 0.1 3.4 ± 0.4 5.3 ± 1.7 Fructose 2.2 ± 0.1 2.8 ± 0.1 2.5 ± 1.3 4.0 ± 1.0 Xylose 1.3 ± 0.2 1.0 ± 0.1 0.6 ± 0.1 0.8 ± 0.1 Galactose 1.0 ± 0.1 0.7 ± 0.2 0.7 ± 0.2 0.6 ± 0.2 The wet chemical analysis (Table 5 ) reveals the presence of glucose, fructose, xylose, and galactose indicating the successful hydrolysis especially of the hemicellulose component of the biomasses studied. The carbohydrates identified are in line with previous findings regarding oak-based hemicelluloses. 45 – 47 These results are confirmed by the HSQC analyses, with the interesting exception of the xylose component in SO samples. While the wet-chemical test shows higher quantities for xylose in SO samples than MT samples, HSQC signal intensity suggests the contrary. The reason for this cannot be delineated precisely on the basis of the data available. More importantly, however, HSQC analyses were able to unveil the presence of additional carbohydrates and derivatives that the wet-chemical test could not target. Via a series of characteristic cross-peaks assigned on the basis of various literature sources 48 – 57 , essentially the same species were identified in all the samples, with variations in relative abundances. SO -derived samples show in HSQC analyses only traces of xylose and methyl glucuronic acid. MT -samples show in HSQC analyses traces in arabinose, whereas this carbohydrate is not clearly detectable in SO samples. Acetylated carbohydrates have been identified in form of acetylated xylose in all samples. This is indicative of the expectable presence of acetylated xylan moieties, especially in MT samples, and thus in line with one of the most famous representatives of hardwood hemicellulose features. HSQC spectra indicate also the presence of alkanoates, originating eventually from waxes present in the hydrolysate. Other weak cross-peaks could be assigned to alkyl residues, eventually stemming from alkylated sugars. Most importantly, SO -derived hydrolysates SOW and SOB contain very weak cross-peaks in the aromatic region that could potentially indicate the presence of pyrogallyl and gallate units, as such presence in tannins, with the latter especially in the typically oak-derived tannic acid. The cross-peak typical for a gallate unit is more pronounced in the SOB sample, whereas here the pyrogallyl peak is hardly detectable. This is a relevant observation since propyl gallate was demonstrated to increase microbial carotenogenesis in the thraustochytrids. 58 The combined NMR analyses do not indicate the presence of lipids, since typical glycerol-derived cross-peaks are absent. Neither amino acids or peptide residues were detected. Samples do contain nitrogen sources, however, as revealed by wet-chemical tests, but contents in amino acids are supposedly too low as that they would exceed the detection threshold of around 1–2% (w/w) typical for NMR analyses. Figure 1 B shows the amount of primary nitrogen source found in the hydrolysate. Nitrogen content is not increased by the action of saccharifying enzymes, with MTB as an exception. The measured amount of nitrogen permits to calculate C/N ratio for the different hydrolysate, being 38 for SOW, 80 for SOB, 34 for MTW and 97 for MTB. C/N ratio is one of the factors involved in the triggering to produce lipids and carotenoids in oleaginous yeasts 7 Given the presence of both carbon and nitrogen sources, the ability of R. toruloides to grow in such media without any supplementation of nutrients, or elimination of possible toxic compounds was directly tested, to further improve the sustainability aspects of the present work in respect to published bioprocesses based on this microbial cell factory and residual biomasses (Table 1 ). 3.3. R. toruloides production of carotenoids from woody hydrolysate Once the presence of fermentable sugars in the hydrolysates was confirmed, the ability of R. toruloides to grow in such media, withstanding possible inhibitory compounds, and to produce carotenoids was tested. As described in Section 2.7, the yeast was directly inoculated into the woody hydrolysate and sampled over time for growth, sugar consumption, and carotenoids production. Figure 4 summarises the obtained results from the growth kinetics on SOW , SOB , MTW , and MTB hydrolysates. MTB is the biomass supporting the highest growth in terms of OD (Fig. 4 D), which is consistent with having the highest initial sugar content among the hydrolysates (Fig. 1 A). While R. toruloides shows a similar growth profile in MTW and SOW (Fig. 4 A and C) considering the initial difference in the provided sugars, growth in SOB hydrolysate resulted to be linear until reaching a plateau (Fig. 4 D), rather than being exponential. Since the NMR analysis of SOB revealed the presence of substructures typical for tannins, above the NMR detection threshold, which is typically around 5% (w/w); it could be speculated that this tannin presence is interfering with yeast productivity. Similarly, terpenes detected in SOB extractives in higher concentration and variety with respect to the other samples by GC-MS analysis (Table S1 ), could have an inhibitory effect. Nevertheless, R. toruloides was able to grow in all cases, consumed the available carbohydrates, despite not completely, and, most importantly, accumulated carotenoids. Specifically, both β-carotene and torulene were detected intracellularly, with the first being the more abundant of the two. The ratio between the two carotenoids in oleaginous yeasts may vary depending not only on strain, but also on the conditions, such as sugar availability, C/N ratio, or light exposure 59 – 62 . The highest production of β-carotene occurred, in light of the above discussed inhibitors, interestingly on SOB hydrolysate after 30 h of growth, reaching 362.7 ± 33.9 mg/L (Fig. 4 B), followed by MTB with 165.3 ± 22.6 mg/L of β-carotene (after 30 h of growth) (Fig. 4 D). The production of torulene was instead observed to be similar across the provided biomass around 24 and 30 h or growth, ranging from 4.5 to 2.5 mg/L. Calculation and comparison of carotenoid yields in the different conditions are summarized in Table 4 , whereas Figure S1 permits to visualize the direct comparison between carotenoids production profiles. These data are in accordance with previous findings disclosing that a C/N ratio of 80, as in SOB hydrolysate, is optimal to maximise β-carotene production over other carotenoids. 62 In addition, as already mentioned, the presence of gallates in SOB hydrolysate can be considered an additional triggering element for carotenogenesis 58 Table 4 Quantitative analysis and yields calculations of β-carotene and torulene production by R. toruloides from SOW , SOB , MTW , and MTB hydrolysates, after 30 h of fermentation. Values are the means ± SD of three independent experiments. Production (mg/L) Yield on initial biomass (10 − 5 g/g) Yield on hydrolysate sugars (g/g) Yield on consumed sugars (g/g) β-carotene Torulene β-carotene Torulene β-carotene Torulene (10 − 2 ) β-carotene Torulene (10 − 2 ) SOW 123.8 ± 10.6 3.6 ± 0.5 83 ± 7 2.4 ± 0.4 1.61 ± 0.20% 4.7 ± 0.8% 2.6 ± 0,4% 7.6 ± 1.6% SOB 362.7 ± 33.8 4.0 ± 0.2 241 ± 23 2.7 ± 0.9 2.62 ± 0.25% 2.9 ± 0.1% 9,2 ± 1,1% 10.1 ± 0.9% MTW 89.9 ± 14.8 1.5 ± 0.2 60 ± 10 1.0 ± 0.1 0.99 ± 0.16% 1.6 ± 0.1% 1.7 ± 0.3% 2.9 ± 0.3% MTB 163.5 ± 22.6 2.6 ± 0.4 109 ± 15 1.7 ± 0.3 1.04 ± 0.16% 1.7 ± 0.3% 1.6 ± 0.3% 2.5 ± 0.5% Table 4 shows, however, that, considering the yield on hydrolysate sugars, SOW hydrolysate can be calculated to represent the best option, after 30 h of growth, for torulene production (0.047 ± 0.008% g/g), and the second best option for β-carotene production (1.61 ± 0.20% g/g). SOB hydrolysate remains the best choice considering β-carotene yields on both initial sugars in the medium and on consumed sugars (Table 4 ). MT -derived samples, in this calculation mode, fall behind the SO -derived ones, for both β-carotene and torulene production. This is consistent with previous data on R. toruloides growth on residual biomasses hydrolysates, where the maximum production of carotenoids was reached between the end of the exponential phase and the entrance of the stationary one. 34 , 63 In case of SOB the presence of a linear growth rather than an exponential one can be seen as indicative of the fact that the cells face hurdles, potentially due to the gallate presence ( vide supra ). Indeed, stressful compounds (such as phenolic ones), or more generally stressful growth conditions, are known to act as a trigger to production of carotenoids, while being detrimental to growth. 8 , 64 Therefore, when developing bioprocesses based on residual lignocellulosic biomasses, a trade-off between these actors has to be considered to maximise both growth and carotenogenesis. In order to investigate whether also components comprised in the extractives could represent inhibitors, dedicated studies in this direction were performed. In fact, such compounds are typically present in woody biomasses (like those valorised in this study), while being limited or absent in other organic / lignocellulosic materials, such as wheat bran, tea or molasses. 3.4. Effect of extractives on the production of carotenoids To study the importance of extractives and/or impurities, MTB and SOB were chosen as biomasses since the numbers and respective amounts of extractives in these bark samples were higher compared to the ones found for the wood samples (Table 2 ). Extractives were removed as described in Section 2.13 and extracted MTB and SOB , i.e. , eMTB and eSOB , respectively, were subjected to enzymatic hydrolysis and SHF following the established procedures. Data obtained from growth in terms of optical density (OD), sugar concentration over time, and carotenoids produced allowed for determining the impact of the presence or absence of extractives in the biomasses. Data are summarised in form of Fig. 6 . Figure 6 shows that the elimination of extractives did not have an impact on enzymatic hydrolysis since the amount of released sugars present in the starting medium is comparable regardless of their presence. We expected the elimination of extractives might improve microbial performance and, in turn, the production of carotenoids. Indeed, eSOB hydrolysate permits for a higher OD compared to SOB , despite a comparable sugar consumption, suggesting that the extractives are responsible of impairing yeast growth. On the other hand, no significant difference can be observed between growth on MTB and eMTB . Surprisingly and independently from growth recovering, the absence of extractives in eSOB and eMTB resulted in a reduced accumulation of carotenoids (Fig. 6CD), compared to SOB and MTB derived carotenoids, implying that the presence of extractives, while being inhibitors, acts as a trigger for carotenogenesis. In fact, the production of carotenoids on eMTB and eSOB hydrolysates is comparable to previous studies whose starting feedstock for R. toruloides do not typically contain significant amount of such extractives ( e.g. wheat bran, tea, molasses) (Table 1 ). Indeed, some of the molecules identified in either SOB or MTB extractives (Table S1 ) are associated with antimicrobial activity, like in the case of lupeol 65 , β-amyrin 66 and benzaldehyde 67 possibly causing a stressful environment leading to carotenogenesis. These observations provide novelty in terms of understanding that extractive components of lignocellulosic biomasses can act as promoting elements for the production of the desired molecule, especially when it is triggered by stressful conditions (like carotenogenesis). These findings are in line with previous literature, which suggest the role of different types of stress in the production of carotenoids by R. toruloides. 8 Furthermore, in terms of biorefinery development, detoxification of the initial biomass is not needed in this case, making the process simpler. These data also suggest that growth and production of carotenoids are not strictly correlated, depending on the environmental conditions. We can thus correlate microbial nutritional needs and biomass composition to the production of the desired compounds, which is definitely a relevant issue in the field of bioprocesses and biorefineries. Further investigations, beyond the scope of this work, might involve genomic and transcriptomic analysis of carotenogenesis related genes in R. toruloides , to better understand the effect of extractives (as a cohort or as single compounds) on cellular behaviour and regulation. 4. Conclusion This study demonstrates the potential of leveraging diverse urban lignocellulosic residues as feedstock for microbial-based biorefineries. The findings here disclosed confirm that the enzymatic hydrolysis effectively releases fermentable sugars from previously underutilized sessile oak and mulberry tree biomasses. R. toruloides exhibited robust performance, growing on these hydrolysates independently from their different sources, and efficiently accumulating carotenoids, including β-carotene and torulene. The observation that natural inhibitors present in the hydrolysates stimulate carotenoid biosynthesis underscores a critical element: challenging biomass components can, in fact, enhance certain desired product formation, optimizing the bioprocess without requiring intensive pre-treatment for inhibitor removal. Interestingly, when extractive components were removed from the biomass, carotenoids production was reduced, being more comparable with previous results from other works using other biomasses. This research contributes to the advancement of a circular bioeconomy by transforming local, readily available waste streams into valuable bio-products. It provides a robust framework for valorising biodiversity and minimizing environmental impact associated with waste disposal. Specifically, this biorefinery approach demonstrates the valorisation of regional woody biomass for the production of high-value pigments and food additives, contributing to a broader understanding of biodiversity valorisation. Future work will focus on further optimizing the bioprocess, and deepening the understanding of inhibitor-carotenogenesis interactions. Furthermore, considering the intrinsic differences between hardwood and softwood ( e.g. , density), which can affect enzymatic hydrolysis efficiency, investigating the effect of different parts of the same plant on the process could be a valuable area to explore. Moreover, to truly "close the loop" in this biorefinery concept, investigations into the valorisation of the remaining lignin-rich residues are paramount. Declarations Competing Interests S.B. declares to be part of the editorial board of Biotechnology for Biofuels and Bioproducts.The authors declare they do not have any further competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Ethics, Consent to Participate, and Consent to Publish not applicable. Funding The authors acknowledge funding under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4 - Call for tender No. 3138 of 16 December 2021, rectified by Decree n.3175 of 18 December 2021 of Italian Ministry of University and Research funded by the European Union – NextGenerationEU. Award Number : Project code CN_00000033, Concession Decree No. 1034 of 17 June 2022 adopted by the Italian Ministry of University and Research, CUP H43C22000530001, Project title “National Biodiversity Future Center - NBFC”. Author Contribution S.B., F.S., G.M.B., M.D., V.T. were involved in Investigation and Data curation.S.B., F.S., V.T., H.L., P.B. were involved in Conceptualization.S.B. was involved in Writing-Original draft preparation.S.B., F.S., V.T., D.P., M.O., H.L., P.B. were involved in Writing-Reviewing and Editing.D.P., M.O., H.L., P.B. were involved in Funding acquisition and Supervision. References Bertacchi, S., Jayaprakash, P., Morrissey, J. P. & Branduardi, P. Interdependence between lignocellulosic biomasses, enzymatic hydrolysis and yeast cell factories in biorefineries. Microbial Biotechnology Preprint at https://doi.org/10.1111/1751-7915.13886 (2021). Gaur, S., Kaur, M., Kalra, R., Rene, E. R. & Goel, M. Application of microbial resources in biorefineries: Current trend and future prospects. Heliyon 10, e28615 (2024). Yogalakshmi, K. N. et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8187172","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":557523241,"identity":"4bbcb0dd-3028-499b-934b-9917b7222f33","order_by":0,"name":"Stefano Bertacchi","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Stefano","middleName":"","lastName":"Bertacchi","suffix":""},{"id":557523243,"identity":"04e3bede-10e6-4121-af65-bf414ea1d3d0","order_by":1,"name":"Francesca Sabatini","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Sabatini","suffix":""},{"id":557523244,"identity":"0947aa44-f07c-4420-b9ef-907efbd4c56a","order_by":2,"name":"Giovanni Maria Bernardini","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Giovanni","middleName":"Maria","lastName":"Bernardini","suffix":""},{"id":557523245,"identity":"a3da7b27-00b9-456f-9718-cb0ae0df15c8","order_by":3,"name":"Matilde Dameri","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Matilde","middleName":"","lastName":"Dameri","suffix":""},{"id":557523248,"identity":"e358a658-aa4f-4e17-9b41-3a59630cdd40","order_by":4,"name":"Veronica Termopoli","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Veronica","middleName":"","lastName":"Termopoli","suffix":""},{"id":557523250,"identity":"15b9c6f7-be4c-4386-af36-fc527b6c5698","order_by":5,"name":"Danilo Porro","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Danilo","middleName":"","lastName":"Porro","suffix":""},{"id":557523253,"identity":"92cee257-3d1f-4995-a85c-16af94ee2468","order_by":6,"name":"Marco Orlandi","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Orlandi","suffix":""},{"id":557523254,"identity":"b0d98dd3-b98f-45aa-819d-dc9d47e49008","order_by":7,"name":"Heiko Lange","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Heiko","middleName":"","lastName":"Lange","suffix":""},{"id":557523256,"identity":"e9bf90b3-94ca-473f-adcf-022d3028d8fd","order_by":8,"name":"Paola Branduardi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYHACxgMMDAdADAMgZubhZwBxDQ4wsOHRg6pFsgGmBY8eFC0gxVBBHFrM2Q8fOPCD4U4+/+zmbRI/91jLGN/IPXjoRsEdBj75BqxaLHvSEg72MDyznHHnWJlkz7N0HrMbeQmHcwye4XSYwYEcgwM8DIcNGG7kmEnwHDgM1JJjANRyGLeW828MDv4BapEHapH8A9RiPIOQFpCZIFuADDNpkC0GEgS0WM54lnBYxuCZgeGNtGJrmQPpPBJn3oC0PONhY0vAHmL8yQcfvqm4YyB3I3njzTcHrO3523OMP+f8uSMn33wAu8OQSFTAg91Z2BWPglEwCkbBKEAFAAzLY+I2ilB5AAAAAElFTkSuQmCC","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":true,"prefix":"","firstName":"Paola","middleName":"","lastName":"Branduardi","suffix":""}],"badges":[],"createdAt":"2025-11-23 18:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8187172/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8187172/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13068-026-02749-3","type":"published","date":"2026-03-24T16:09:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":97870616,"identity":"b366f634-3f09-4e72-b03c-cbb30c0eda4d","added_by":"auto","created_at":"2025-12-10 10:18:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5531378,"visible":true,"origin":"","legend":"","description":"","filename":"MaintextBertacchietal.docx","url":"https://assets-eu.researchsquare.com/files/rs-8187172/v1/be1e80067cc28f5429629112.docx"},{"id":97900546,"identity":"29b2b3ec-50f1-496a-ae82-c7cfde1c4c6e","added_by":"auto","created_at":"2025-12-10 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15:38:41","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":44821,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8187172/v1/e3ad303de950ca1110c97ac6.png"},{"id":97898626,"identity":"4842f836-856d-467f-bf5e-c1888bf3eb8e","added_by":"auto","created_at":"2025-12-10 15:39:22","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":38063,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8187172/v1/737c82b37209759ad701645b.png"},{"id":97899892,"identity":"b2949ba3-c6ca-4946-bd6a-562c217c0a74","added_by":"auto","created_at":"2025-12-10 15:45:02","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":176966,"visible":true,"origin":"","legend":"","description":"","filename":"978514aba8714f90aafb2d911dff62191structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8187172/v1/20bfcfba2c97fe9494519cc0.xml"},{"id":97870625,"identity":"6900c654-29f7-4a63-b61b-c46db1e5cc4b","added_by":"auto","created_at":"2025-12-10 10:18:49","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":192368,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8187172/v1/16cce76be64d50e50881b24f.html"},{"id":97870609,"identity":"13666c38-f87b-4718-a6ed-8eb7770e01cb","added_by":"auto","created_at":"2025-12-10 10:18:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":133826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 1:\u003c/strong\u003e Total reducing sugars released by enzymatic hydrolysis of the different woody biomass (A) and corresponding primary nitrogen sources (B). Values are the means ± SD of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8187172/v1/b638574fd7570714281b254f.png"},{"id":97870610,"identity":"fef3fc7b-27be-4d24-8a32-6e618b316d74","added_by":"auto","created_at":"2025-12-10 10:18:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":477966,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 2:\u003c/strong\u003e SEM images of grounded biomass particles: (A) \u003cstrong\u003eSOW\u003c/strong\u003e; (B) \u003cstrong\u003eSOB\u003c/strong\u003e; (C) \u003cstrong\u003eMTW\u003c/strong\u003e; and (D) \u003cstrong\u003eMTB\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8187172/v1/eea40751186600ed6a512ba3.png"},{"id":97899874,"identity":"7cbf966c-5fba-4df5-b3a2-19f7f0de117f","added_by":"auto","created_at":"2025-12-10 15:45:00","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":397032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 3:\u003c/strong\u003e \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HSQC spectra of (A) \u003cstrong\u003eSOW\u003c/strong\u003e full spectra; (B) \u003cstrong\u003eSOW\u003c/strong\u003e partial aliphatic \u0026amp; numeric region; (C) \u003cstrong\u003eSOB\u003c/strong\u003e partial aliphatic \u0026amp; numeric region; (D)\u003cstrong\u003e MTW\u003c/strong\u003e partial aliphatic \u0026amp; numeric region; (E) \u003cstrong\u003eMTB\u003c/strong\u003e partial aliphatic \u0026amp; numeric region; and (F) \u003cstrong\u003eMTB\u003c/strong\u003e partial aliphatic \u0026amp; numeric region after 72 hours.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8187172/v1/cdfdb57a8f33bfd7b4000fdb.jpeg"},{"id":97870611,"identity":"a2d9be5e-5380-4cb5-b3cc-9e9edbe56cfa","added_by":"auto","created_at":"2025-12-10 10:18:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":151820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4:\u003c/strong\u003e Fermentation profile and carotenoids production by \u003cem\u003eR. toruloides\u003c/em\u003e on (A) \u003cstrong\u003eSOW\u003c/strong\u003e, (B) \u003cstrong\u003eSOB\u003c/strong\u003e, (C) \u003cstrong\u003eMTW\u003c/strong\u003e and (D) \u003cstrong\u003eMTB\u003c/strong\u003ehydrolysates. Values are the means ± SD of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8187172/v1/d01565ff7e1c394c6d05ac3d.png"},{"id":97898653,"identity":"5d354876-5f88-4d38-a47e-af6b0bc0530a","added_by":"auto","created_at":"2025-12-10 15:39:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":285651,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 6:\u003c/strong\u003e Fermentation profiles of \u003cem\u003eR. toruloides\u003c/em\u003eduring growth on (A) \u003cstrong\u003eeSOB\u003c/strong\u003e and (B) \u003cstrong\u003eeMTB\u003c/strong\u003e hydrolysate. In these panels data regarding the fermentation on \u003cstrong\u003eSOB\u003c/strong\u003e or \u003cstrong\u003eMTB\u003c/strong\u003e hydrolysate are repeated from Figure 4 in order to highlight differences. Production of carotenoids from (C) \u003cstrong\u003eeSOB\u003c/strong\u003e and (D) \u003cstrong\u003eeMTB\u003c/strong\u003e. Values are the means ± SD of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8187172/v1/2f2187b1a37d00823fe8655d.png"},{"id":105755903,"identity":"363a6abd-fcfe-4624-bbfd-d82c1b27e782","added_by":"auto","created_at":"2026-03-30 16:32:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3141322,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8187172/v1/31346a22-fd54-4486-a5fa-8152c86c0a97.pdf"},{"id":97900094,"identity":"7a602d06-db39-49f2-8960-d9f10070efa8","added_by":"auto","created_at":"2025-12-10 15:45:13","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":53625,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformationBertacchietal.docx","url":"https://assets-eu.researchsquare.com/files/rs-8187172/v1/8e4106f2d74100ddc5129f81.docx"}],"financialInterests":"Competing interest reported. S.B. declares to be part of the editorial board of Biotechnology for Biofuels and Bioproducts.\nThe authors declare they do not have any further competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.","formattedTitle":"Rhodotorula toruloides for carotenoid production using waste hardwood biomass","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe exploration of diverse biological feedstocks and microorganisms is crucial for establishing efficient and environmentally friendly biorefinery and bioconversion pathways \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Woody biomasses, derived from pruning and forestry, agricultural wastes, as well as dedicated energy crops, constitute a largely untapped resource for biorefineries due to their prevalent lignocellulosic composition. These renewable materials offer in form of their comprised polymers cellulose, hemicelluloses, and lignins valuable alternatives to fossil resources for the production of fuels, chemicals, and high-value bioproducts within the bioeconomy scenario \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe integration of microbial biotechnology into biorefinery processes can be a key factor to unlock the full valorisation potential of woody biomasses. Enzymatic hydrolysis enables the release of fermentable pentose and hexose sugars from the cellulose and hemicellulose fractions, providing carbon sources for microbial growth and biotransformation.\u003c/p\u003e\u003cp\u003eAmong the diverse microbial candidates for lignocellulosic biomass valorisation, the oleaginous yeast \u003cem\u003eRhodotorula\u003c/em\u003e (\u003cem\u003eRhodosporidium\u003c/em\u003e) \u003cem\u003etoruloides\u003c/em\u003e has gained considerable attention. This yeast exhibits a remarkable ability to metabolize a wide range of sugars derived from lignocellulosic hydrolysates, \u003cem\u003ei.e.\u003c/em\u003e, both pentose and hexose sugars, and has demonstrated tolerance to various inhibitory compounds that can arise during biomass pretreatment \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Furthermore, \u003cem\u003eR. toruloides\u003c/em\u003e is a lipidogenic yeast and natural producer of valuable carotenoid pigments, including β-carotene and torulene. This class of compounds features significant antioxidant and provitamin A activity, and colouring properties with applications in food, feed, cosmetic, and pharmaceutical industries \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The global market for carotenoids continues to grow (USD 1.48\u0026nbsp;billion in 2023, projected to grow at a CAGR of 3.5% to 2030), driven by increasing consumer demand for natural and health-promoting ingredients, as well as bio-based colorants, with ꞵ-carotene products accounting for a 24.7% revenue share in 2023 \u003csup\u003e13\u003c/sup\u003e. The torulene market, although smaller, is attracting an increasing interest due to its biological properties and potential health benefits \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eDespite the growing insight into the capabilities of \u003cem\u003eR. toruloides\u003c/em\u003e in the context of lignocellulosic biomass valorisation, research that specifically investigates the utilization of novel woody feedstocks from local biodiversity for carotenoid production is yet underexplored. While several studies have examined \u003cem\u003eR. toruloides\u003c/em\u003e growth on conventional agricultural residues (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the potential of underutilized woody forestry residues deserves further investigation. Recent studies in the literature deploying woody hydrolysate as possible substrates for \u003cem\u003eR. toruloides\u003c/em\u003e, are either centred on lipid rather than carotenoid production, or they report the introduction of additional carbon and/or nitrogen sources \u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Additionally, most of the recent publications on the topic are focused on the valorisation of by-products from pruning, mainly of olive trees\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e or vines \u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, whereas urban pruning residues are exploited mainly for energy or chemical production \u003cem\u003evia\u003c/em\u003e physical and/or chemical treatments \u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Thus, there is a lack of studies on the valorisation of such urban residues by microbial action, beyond composting or anaerobic digestion\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Therefore, since the current use of such residues for low value products (\u003cem\u003ei.e.\u003c/em\u003e energy), this study aims to providing alternative valorisation for these lignocellulosic biomasses of urban origin to obtain a product with an higher value (\u003cem\u003ei.e.\u003c/em\u003e carotenoids).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExamples from literature for the production of carotenoids from residual biomasses by the use of \u003cem\u003eR. toruloides\u003c/em\u003e strains, with a focus on the comparison between productivities obtained. \u003cb\u003eSOW\u003c/b\u003e, \u003cb\u003eSOB\u003c/b\u003e, \u003cb\u003eMTW\u003c/b\u003e, and \u003cb\u003eMTB\u003c/b\u003e, as disclosed in the text, represent wood (\u003cb\u003eW\u003c/b\u003e) and bark (\u003cb\u003eB\u003c/b\u003e) of sessile oak (\u003cb\u003eSO\u003c/b\u003e) and mulberry tree (\u003cb\u003eMT\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR. toruloides\u003c/p\u003e\u003cp\u003estrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eBiomass\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAdded N source\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStarting sugars\u003c/p\u003e\u003cp\u003e(g/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTime (h)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCarotenoid\u003c/p\u003e\u003cp\u003eProduction (mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eCarotenoid productivity (mg/L/h)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDSM 4444\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDefatted waste wheat bran\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eBiological (Cellic\u0026reg;\u003c/p\u003e\u003cp\u003eCTec 3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eYeast extract, ammonium sulfate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e144\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e180\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003csup\u003e27\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eACCC20341\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTea waste\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eChemical (Sulfuric acid)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eYeast extract, ammonium sulfate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e95.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e180\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.875\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003csup\u003e28\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC23\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSynthetic lignocellulosic-like medium\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eYeast extract, ammonium sulfate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e120\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e144\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003csup\u003e29\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eL/24-26-1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSugarcane molasse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eChemical (Sulfuric acid)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eYeast extract, ammonium sulfate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e168\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e21.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003csup\u003e30\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNCIM 3547\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNeem oilseed cake\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eChemical (Microwave mild acid hydrolysis)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eYeast extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e25.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e60.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003csup\u003e31\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDSM 4444\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSpent coffee grounds\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eBiological (β-glucosidase Viscozyme)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eYeast extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e23.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003csup\u003e32\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDSM 4444\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eMTW\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMTB\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSOW\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSOB\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eeMTB\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eeSOB\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eBiological (Novozymes NS22119)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9.1\u003c/p\u003e\u003cp\u003e15.7\u003c/p\u003e\u003cp\u003e7.7\u003c/p\u003e\u003cp\u003e13.8\u003c/p\u003e\u003cp\u003e13.3\u003c/p\u003e\u003cp\u003e12.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e89.9\u003c/p\u003e\u003cp\u003e163.5\u003c/p\u003e\u003cp\u003e123.8\u003c/p\u003e\u003cp\u003e362.7\u003c/p\u003e\u003cp\u003e10.6\u003c/p\u003e\u003cp\u003e25.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.9\u003c/p\u003e\u003cp\u003e5.5\u003c/p\u003e\u003cp\u003e4.1\u003c/p\u003e\u003cp\u003e12.1\u003c/p\u003e\u003cp\u003e0.35\u003c/p\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003eThis study\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/strong\u003e\u003cp\u003eExamples from literature for the production of carotenoids from residual biomasses by the use of \u003cem\u003eR. toruloides\u003c/em\u003e strains, with a focus on the comparison between productivities obtained. \u003cb\u003eSOW\u003c/b\u003e, \u003cb\u003eSOB\u003c/b\u003e, \u003cb\u003eMTW\u003c/b\u003e, and \u003cb\u003eMTB\u003c/b\u003e, as disclosed in the text, represent wood (\u003cb\u003eW\u003c/b\u003e) and bark (\u003cb\u003eB\u003c/b\u003e) of sessile oak (\u003cb\u003eSO\u003c/b\u003e) and mulberry tree (\u003cb\u003eMT\u003c/b\u003e). \u003cb\u003eeMTB\u003c/b\u003e and \u003cb\u003eeSOB\u003c/b\u003e represent the corresponding biomasses without the extractive components.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eThis study aims consequently at addressing the potential of urban pruning/forestry residues in the form of sessile oak (\u003cem\u003eQuercus petraea\u003c/em\u003e, \u003cb\u003eSO\u003c/b\u003e) and mulberry tree (\u003cem\u003eMorus alba\u003c/em\u003e, \u003cb\u003eMT\u003c/b\u003e) biomasses, sourced from an urban park within the Milan metropolitan area, as substrates for \u003cem\u003eR. toruloides\u003c/em\u003e growth and carotenoid production.\u003c/p\u003e\u003cp\u003eThe initial steps of this work involve the hydrolysis of the residual biomasses to obtain growth media for the yeast, followed by the analysis of the resulting sugar and nitrogen concentrations from both wood (\u003cb\u003eW\u003c/b\u003e) and bark (\u003cb\u003eB\u003c/b\u003e) of sessile oak and mulberry tree, \u003cem\u003ei.e.\u003c/em\u003e, \u003cb\u003eSOW\u003c/b\u003e, \u003cb\u003eSOB\u003c/b\u003e, \u003cb\u003eMTW\u003c/b\u003e, and \u003cb\u003eMTB\u003c/b\u003e. Furthermore, the woody hydrolysates were presented to \u003cem\u003eR. toruloides\u003c/em\u003e in a separated hydrolysis and fermentation (\u003cb\u003eSHF\u003c/b\u003e) setup. Differences in carotenoid production during fermentation time were analysed, with particular attention to identify components of the woody biomass that might trigger or inhibit carotenoid biosynthesis.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. General information\u003c/h2\u003e\u003cp\u003eAll chemicals used were analytical grade. Reagent grade water was provided by a Milli-Q\u0026reg; Integral 5 purification system (Merck KGaA, Darmstadt, Germany). Dichloromethane, toluene, ethanol, ethyl acetate and acetone (GC-MS grade) were purchased from Carlo Erba (Cornaredo, Italy). Sulphuric acid (95\u0026ndash;97%) was obtained from Merck KGaA (Darmstadt, Germany). Acetonitrile (LC-MS grade) was supplied from VWR International SrL (Milan, Italy). Pure standards of β-carotene and caffeine were obtained from Sigma-Aldrich (St Louis, MO, USA), while the standard of torulene was provided by CaroteNature (M\u0026uuml;nsingen, Switzerland).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Biomass collection and preparation\u003c/h2\u003e\u003cp\u003eThe residual woody biomasses used in this study are sessile oak (\u003cem\u003eQuercus petraea\u003c/em\u003e) wood (\u003cb\u003eSOW\u003c/b\u003e) and bark (\u003cb\u003eSOB\u003c/b\u003e), as well as mulberry tree (\u003cem\u003eMorus alba\u003c/em\u003e) wood (\u003cb\u003eMTW\u003c/b\u003e) and bark (\u003cb\u003eMTB\u003c/b\u003e). These materials were collected as leftovers from the maintenance of Parco della Besozza (Pioltello, Lombardy), an urban park in the metropolitan area of Milan (Italy). These biomasses were used as obtained, \u003cem\u003ee.g.\u003c/em\u003e, in the form a conventional urban pruning/forestry workflow that produced them in an only roughly separated form between bark and wood. Consequently, both starting materials mutually contain residues of the other component, \u003cem\u003ei.e.\u003c/em\u003e, bark or wood. Importantly, these biomasses were applied in this study without any upstream detoxification step, yielding the possibility to explore eventual inhibiting factors. The biomasses were dried and stored at room temperature till processing. Woody biomasses were mechanically grounded to a final particle size of less than 1 mm. The resulting powdered biomass was used for all the steps of treatment and analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Scanning electron microscope (SEM) analysis of grounded biomasses\u003c/h2\u003e\u003cp\u003eSEM-EDS observations were performed at the Platform of Microscopy of the University of Milano-Bicocca (PMiB) with a field emission gun (FEG) SEM Zeiss Gemini 500, operating at 3 and 5 keV and equipped with a Bruker QUANTAX integrated wave-dispersive/energy-dispersive (WDS/EDS) system. Metallization was carried out using an Edwards Sputter Coater S150B, with Au/Pd target (70% Au/30%Pd) in Ar atmosphere, current of 40 mA, voltage 1 kV, suttering time 1 min, and depositing a 10 nm thick Au/Pd layer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Removal of extractives from grounded biomasses\u003c/h2\u003e\u003cp\u003eWood or bark powder was placed into a crucible glass filter, pore size III, for subsequent solvent extraction using a standard 100 mL Soxhlet extractor. The first extraction utilized 150 mL of dichloromethane (DCM) for 6 h, followed by a second extraction using 150 mL of a 1:1 (v/v) toluene/ethanol (tol/EtOH) mixture for 12 h. The final extraction used 150 mL of acetone for 6 h. The extracts were subsequently isolated by removing the solvents \u003cem\u003ein vacuo\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Determination of acid-insoluble and acid-soluble lignin content through Klason extraction\u003c/h2\u003e\u003cp\u003eFor the determination of acid-insoluble lignin, a NREL procedure was adopted (Lin and Dence 1992). Approximately 100 mg of milled wood or bark was carefully weighed into a test tube. To this, 2 mL of 72% (w/v) sulfuric acid (H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) solution was added at room temperature, with intermittent stirring over a 2 h. The resulting solution was then diluted with deionized water to achieve a 3% (w/v) H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e concentration. Then, the solution was transferred to a sealed flask and subjected to heating at 120\u0026deg;C and 2 bar for 7 min using an autoclave (LaborAutoklav, Certoclav, Austria). The solution was filtered through a crucible of size M, washed with deionized water until a neutral pH was attained, and the crucibles were placed in an oven at 105\u0026deg;C for 12 h to fully dry the sample. The determination of acid-insoluble lignin was performed gravimetrically.\u003c/p\u003e\u003cp\u003eFor the assessment of acid-soluble lignin, the filtrate obtained from the acid-insoluble lignin procedure was diluted to 100 mL using deionized water. The quantification of acid-soluble lignin was performed by calculating UV absorbance at 205 nm, utilizing an extinction factor of 113 L∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e∙cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was chosen according to literature for hardwoods (Lin and Dence 1992).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Enzymatic hydrolysis of woody biomasses.\u003c/h2\u003e\u003cp\u003eEnzymatic hydrolysis of the woody biomasses was performed using the enzyme cocktail NS22119, kindly provided by Novonesis (Novonesis A/S, Copenhagen, Denmark). NS22119 contains a wide range of carbohydrases, including cellulase, arabinase, pectinase, β-glucanase, hemicellulase, and xylanase from \u003cem\u003eAspergillus aculeatus\u003c/em\u003e, as described by the producer. As a first step, 15% of biomass (w/v) was autoclaved at 121\u0026deg;C for 1 h to both sterilize and mildly pre-treat the biomass. Afterward, NS22119 enzyme mix (11.9% w/w\u003csub\u003e\u003cem\u003ebiomass\u003c/em\u003e\u003c/sub\u003e) was added to the biomass and incubated at 50\u0026deg;C in a water bath under mild agitation (105 rpm) for 6 h. At the end of the process, the hydrolysates were centrifuged (8000 rpm, 10 min) to precipitate the insoluble components and collect the supernatant to be used as growth medium for \u003cem\u003eR. toruloides\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Microbial strain, media and growth conditions\u003c/h2\u003e\u003cp\u003e\u003cem\u003eRhodotorula (Rhodosporidium) toruloides\u003c/em\u003e DSM 4444 was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, GmbH) and kept in cryotubes at \u0026minus;\u0026thinsp;80\u0026deg;C in 20% glycerol (v/v). The composition of the medium (YPD) for the pre-inoculum was as follows: 20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose. Yeast extract and peptone was purchased from Biolife Italia S.r.l. (Milan, Italy). All other reagents were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). After plating on YPD, a pre-inoculum was run in rich medium until the stationary phase. Then, cells were inoculated at 0.25 OD in shake flasks at 30\u0026deg;C and 160 rpm in SHF setting using woody hydrolysates. Growth was measured in terms of optical density (OD) at 600 nm. Samples (1 mL) were collected during the growth and centrifuged (8000 rpm, 5 min). The supernatant was used for sugar quantification, whereas the cellular pellet was used for carotenoid extraction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Intracellular carotenoids extraction\u003c/h2\u003e\u003cp\u003eAcetone was used for extracting carotenoids from \u003cem\u003eR. toruloides\u003c/em\u003e cells as described elsewhere (Bertacchi et al. 2020). In brief, 1 mL of cells was collected and harvested by centrifugation at 10000 rpm for 7 min, and the pellet was then resuspended in 1 mL acetone and broken using glass beads by thorough agitation with a FastPrep-24\u0026trade; (MP Biomedicals, LLC, Santa Ana, CA, USA) with 3 cycles of 30 sec each at 4\u0026deg;C. Carotenoids were thus extracted in the acetone phase, collected in the supernatant by centrifugation and stored at -20\u0026deg;C. The extraction was repeated with a new aliquot of acetone until the biomass turned colourless.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Characterization of woody hydrolysate using dinitrosalicylic (DNS) test\u003c/h2\u003e\u003cp\u003eThe dinitrosalicylic (DNS) test was used to determine the total reducing sugar content of the obtained hydrolysed. An optimized and miniaturized DNS protocol was deployed.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e DNS reagent consists of: 10 g/L 3,5-dinitrosalicylic acid (Sigma-Aldrich), 16 g/L NaOH (Merck), 30 g/L potassium sodium tartrate (Carlo Erba). For quantification, a glucose calibration curve (30\u0026ndash;0.6 g/L) was prepared. In each tube, 190 \u0026micro;L of the DNS reagent and 10 \u0026micro;L of sample were inserted and then vortexed. The tube was heated at 100\u0026deg;C for 2 min and then cooled to room temperature. Following heat treatment, all samples were transferred in 96-well plates and analysed at 531 nm in a multiplate reader (VICTOR X3, PerkinElmer). Specific sugars (glucose, fructose, sucrose, xylose, galactose) were quantified by using the spectrophotometric enzymatic kits K-SUFRG, K-XYLOSE and K-ARGA (Megazyme, Southern Cross Rd, Bray, Co. Wicklow, Ireland), following the manufacturer\u0026rsquo;s instructions. For this purpose, samples were treated by the addition of 20 g/L of polyvinylpolypyrrolidone (PVPP), shaken vigorously for 5 min and then filtered. Nitrogen content in the form of primary amines was quantified by the use of PANOPA enzymatic kit (Megazyme).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Determination of yields and C/N ratio\u003c/h2\u003e\u003cp\u003e\u0026ldquo;Yield of carotenoids on initial biomass\u0026rdquo; was calculated as the ratio between the carotenoids production and the amount of biomass used in the enzymatic hydrolysis. Similarly, \u0026ldquo;yield on hydrolysate sugars\u0026rdquo; or \u0026ldquo;yield on consumed sugars\u0026rdquo; have as denominator the amount of sugars in the hydrolysate provided to cells as growth medium or the amount of sugars effectively consumed after 30 h of growth, respectively.\u003c/p\u003e\u003cp\u003eC/N ratio of hydrolysate was calculated as the ratio between the carbon content in the sugars measured by DNS assay and the measured primary nitrogen source. The carbon content was calculated by multiplying total reducing sugars amount by 0.4, as 40% of a glucose and fructose molecule is carbon (and most of the sugars in the media are either glucose or fructose).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Characterization of woody hydrolysate using \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH nuclear magnetic resonance (NMR) spectroscopy\u003c/h2\u003e\u003cp\u003eTo a 500 \u0026micro;L aliquot of the hydrolysate solution were added 100 \u0026micro;L D\u003csub\u003e2\u003c/sub\u003eO as lock-triggering solvent. A Bruker 400 MHz spectrometer controlled \u003cem\u003evia\u003c/em\u003e TopSpin 4.1.4 with a 5 mm double resonance broadband inverse probe was used to acquire \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra at 30\u0026deg;C. The Bruker zgesgp pulse sequence was used with NS\u0026thinsp;=\u0026thinsp;48, applying the presaturation method for water signal suppression. MestreNova Version 9.0.1 (Mestrelab Research S.L.) was used for data processing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Characterization of woody hydrolysate using \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC heteronuclear single quantum coherence (HSQC) analysis\u003c/h2\u003e\u003cp\u003eTo a 500 \u0026micro;L aliquot of the hydrolysate solution 100 \u0026micro;L D\u003csub\u003e2\u003c/sub\u003eO were added as lock-triggering solvent. A Bruker 400 MHz spectrometer controlled \u003cem\u003evia\u003c/em\u003e TopSpin 4.1.4 with a 5 mm double resonance broadband inverse probe was used to acquire HSQC spectra at 30\u0026deg;C. The Bruker hsqcetg pulse program in the DQD acquisition mode was applied with NS\u0026thinsp;=\u0026thinsp;64; TD\u0026thinsp;=\u0026thinsp;2048 (F2) and 512 (F1); SQ\u0026thinsp;=\u0026thinsp;12.9869 ppm (F2) and 164.9996 ppm (F1); O2 (F2)\u0026thinsp;=\u0026thinsp;2601.36 Hz and O1 (F1)\u0026thinsp;=\u0026thinsp;7799.05 Hz; D1\u0026thinsp;=\u0026thinsp;2 s; CNST2 \u003csup\u003e1\u003c/sup\u003eJ(C-H)\u0026thinsp;=\u0026thinsp;145; and acquisition time F2 channel\u0026thinsp;=\u0026thinsp;197.0176 ms and F1 channel\u0026thinsp;=\u0026thinsp;15.4164 ms. For each sample, the pulse length of the 90\u0026deg; P1 high-power pulse was optimized. MestreNova Version 9.0.1 (Mestrelab Research S.L.) was used for data processing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13. Characterization of wood and bark extracts using gas chromatography coupled with mass spectrometry (GC/MS)\u003c/h2\u003e\u003cp\u003eGas Chromatographic-mass spectrometric analyses of the dried extractives were performed by re-dissolving a dried sample of the extracts in 500 \u0026micro;L of dichloromethane. The chromatographic separations were performed using a Shimadzu GCMS QP2020NX (Shimadzu Corporation, Kyoto, Japan) equipped with Shimadzu autosampler AOC20i. An SH-Rxi-5ms fused silica capillary column (stationary phase (5%-Phenyl)-methylpolysiloxane, 30 m x 0.25 mm i.d., 0.25 \u0026micro;m, Shimadzu Corporation, Kyoto, Japan) was used as stationary phase, and He (UHP grade) as carrier gas. The system was operated in \u0026lsquo;linear velocity mode\u0026rsquo; with a starting pressure of 100 kPa, 280\u0026deg;C injection temperature, and 280\u0026deg;C interface temperature. The injection volume was 2 \u0026micro;L, the injection port operated in splitless mode. The temperature program was set as follows: the initial temperature of 50\u0026deg;C was held for 1 min, then increased at a rate of 10\u0026deg;C min\u003csup\u003e˗\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e to a 280\u0026deg;C, which was maintained for 15 min. The MS operated in electron ionization mode (EI) at 70 eV, acquiring in full-scan mode in the \u003cem\u003em/z\u003c/em\u003e range of 50\u0026ndash;500. LabSolutions\u0026ndash;GCMS Version 4.54 software (Shimadzu Corporation) was used as system control, instrument management and data acquisition. Substances were identified using NIST MS Search, version 2.4 (2020).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14. Carotenoids quantification using flow injection analysis coupled by mass spectrometry (FIA/MS)\u003c/h2\u003e\u003cp\u003eFollowing acetone-based extraction of carotenoids described in Section 2.8 of the Materials and Methods, quantitative analysis of β-carotene and torulene was performed on the resulting extracts. β-carotene and torulene were used as analytical standard and caffeine as internal standard (10 ppm) to construct five-point calibration curves (concentration range 0.01-1 ppm for torulene, 0.5\u0026ndash;20 ppm for β-carotene). The calibration curves were obtained in flow injection analysis (FIA) mode, which was applied even for the analysis of the extracts in acetone, previously centrifuged at 1500 rpm for 5 min. A binary LC 20AT pump, coupled with an SPD M20A UV system and a 2010 EV single-quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source was used. All modules are Shimadzu Corporation (Kyoto, Japan). The mobile phase was set at 100% acetonitrile, with a flow rate of 0.3 mL/min, and an injection volume of 20 \u0026micro;L. The operating conditions of the ESI-MS system were as follows: nebulizer gas (N\u003csub\u003e2\u003c/sub\u003e, purity\u0026thinsp;\u0026gt;\u0026thinsp;98%) with flow rate of 1.5 mL/min, curtain desolvation line (CDL) at 240\u0026deg;C and heat block (HB) at 230\u0026deg;C, capillary voltage 2.5 kV. Acquisition of MS spectra was performed in positive ion mode in selected ion monitoring (SIM), observing fragments at m/z 195 for caffeine, 534 for torulene, and 536 for β-carotene. LCMS solution Version 3.30 268 software (2002\u0026ndash;2005; Shimadzu Corporation) was used for instrument management and data acquisition.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Characterization of woody biomass\u003c/h2\u003e\u003cp\u003eThe complex nature of residual woody biomass is a well-known initial hurdle in defining its application in a microbial-based biorefinery \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Additionally, the intrinsic biodiversity, in terms of plant species and parts, is key to highlight differences in carotenoids production possible arising from the procedure steps as hydrolysis and fermentation (\u003cb\u003eSHF\u003c/b\u003e). For these reasons, the composition of the biomass selected for this study, \u003cem\u003ei.e.\u003c/em\u003e, sessile oak wood (\u003cb\u003eSOW\u003c/b\u003e) and bark (\u003cb\u003eSOB\u003c/b\u003e), as well as mulberry tree wood (\u003cb\u003eMTW\u003c/b\u003e) and bark (\u003cb\u003eMTB\u003c/b\u003e) was initially characterized. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarises the main components constituting these lignocellulosic biomasses, in terms of the sugar-fraction vs. the lignin content, and considering the extractable compounds from the three solvent consecutive extractions.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComposition of biomasses used in this study. Cellulose \u0026amp; hemicellulose [% (w/w)] are calculated as the difference of the sum of lignin (acid soluble), lignin (acid insoluble) and total extractives. Total extractives [% (w/w)] is given by the sum of DCM, EtOH/tol and acetone consecutive extractions.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBiomass component\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSOW\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSOB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMTW\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMTB\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCellulose \u0026amp; hemicellulose [% (w/w)]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e61\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e59\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e53\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLignin (acid soluble) [% (w/w)]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLignin (acid insoluble) [% (w/w)]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e35\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal extractives [% (w/w)]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eOverall, the delineated compositions are in line with what is expected from hardwoods, with less cellulose contents in the barks \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Bulk sugar contents are rather similar, both between the wood components and the bark ones.\u003c/p\u003e\u003cp\u003eThe analysis of the lignin content of the various biomasses (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) shows values that seem high for hardwood species. It can be assumed, based on literature data (K\u0026ouml;gel-Knabner 2021), that a low percentage of the determined insoluble Klason lignin content is actually comprised of humins, known to form upon sugar degradation during Klason analysis and co-precipitating with the insoluble lignin. Interesting is the fact that in the barks is found more lignin than in the bulk. This must be explained by the residual wood in the bulk bark material and the presence of suberins that behave eventually similar to the lignin in the applied NREL procedure \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eDetailed analysis of the various extractives obtained was performed using gas-chromatography coupled to mass spectrometry to provide an in-depth characterization of their composition. Indeed, rather scant information is reported in the literature on the composition of the trees, wood and bark under study. The results, detailed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, show the expectable range of bioactive small molecules, with a slightly richer composition found for two bark samples, \u003cem\u003ei.e.\u003c/em\u003e, \u003cb\u003eSOB\u003c/b\u003e and \u003cb\u003eMTB\u003c/b\u003e. Several molecular classes of compounds have been identified in the extracts, mostly fatty acids and sterols, but also marker species belonging to other chemical classes. Mulberry tree resulted to be richer in extractives than sessile oak, showing high amount and variety of fatty acids, phenolic compounds, vitamins and coumarins especially in the bark. The three isomers of tocopherol (δ-tocopherol, γ-tocopherol, α-tocopherol), an antioxidant compound known as vitamin E, has been already observed in mulberry fruit and bark \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, as well as coumarins especially in leaves \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e but not those detected in our study as umbelliferone, methyl ostruthin, ostruthin and esculetin typical of \u003cem\u003eumbelliferae\u003c/em\u003e family of plants. As regards sessile oak, \u003cb\u003eSOW\u003c/b\u003e profile is particularly poor while \u003cb\u003eSOB\u003c/b\u003e interestingly presents the wider variety of terpenoids of all the four samples. The identified terpenes belong to the family of diterpenes (copalol), tritepenes (β-amyrone, 24-norursa-3,12-diene, lupeol, β-amyrin, glutinol, copalol and simiarenol) and sesquiterpenes (Humulenol-II). SOB extractives show also the unique presence of alcohols, alkenes and other compounds such as benzaldehyde and benzaldehyde diethylacetal, compared to the other biomasses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Enzymatic hydrolysis of woody biomasses\u003c/h2\u003e\u003cp\u003eTo unlock the potential of lignocellulosic biomasses in microbial-based biorefineries, an enzymatic cocktail able to release both hexose and pentose into the liquid phase sugars is commonly used. As described in Section 2.6, powdered biomasses, \u003cem\u003ei.e.\u003c/em\u003e, \u003cb\u003eSOW\u003c/b\u003e, \u003cb\u003eSOB\u003c/b\u003e, \u003cb\u003eMTW\u003c/b\u003e, and \u003cb\u003eMTB\u003c/b\u003e, were subjected to enzymatic hydrolysis. Data reported in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA clearly show the effect of the action of such an enzymatic cocktail in terms of total reducing sugars, with an increase in the carbohydrates released from the sole pre-treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSpecifically, given the same initial amount of biomass processed per volume (15% w/v), data show that \u003cb\u003eMTB\u003c/b\u003e is the raw material providing the highest amount of released sugars (16.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 g/L), whereas on \u003cb\u003eMTW\u003c/b\u003e basis the lowest content is obtained, reaching approx. only 50% of that seen for the \u003cb\u003eMTB\u003c/b\u003e sample (8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 g/L). Interestingly, the bark moiety provided also for the \u003cb\u003eSO\u003c/b\u003e-biomass consistently more sugars compared to the corresponding wood, \u003cem\u003ei.e.\u003c/em\u003e, \u003cb\u003eSOW\u003c/b\u003e. This fact can be related to the different structural characteristics of bark with respect to wood. Scanning electron microscopy (SEM) imagines collected on the various biomasses after grounding show that the bark samples seem slightly more porous, exposing thus a larger surface for the enzymes to work on (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Nevertheless, these data are in contrast with previous research suggesting a negative impact of bark presence towards enzymatic saccharification, using as biomass source residues of spruce, birch, and douglas-fir \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn order to investigate the composition of the carbohydrate mixture in the hydrolysates of the various biomasses, \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HSQC analyses were performed alongside quantification by wet-chemical assessment by the use of enzymatic assays of most of the sugars identified. Tables\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e5\u003c/span\u003e report the results obtained in the assays, whereas Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the HSQC spectra obtained for the various hydrolysates.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCarbohydrates and inhibitors identified in hydrolysates after 6 hours of the biomasses used in this study by means of \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HSQC analyses (\u003cem\u003eHSQC\u003c/em\u003e) and. A full list of identified signals and their respective shifts is given in the Supporting Information.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCarbohydrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ecolour code\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSOW\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSOB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMTW\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMTB\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArabinose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFructose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGalactose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlucose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMannose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethyl gluconoric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eXylose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOther compounds\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlkanoyl residues\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlkyl residues\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGallate residues\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003etraces\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePyrogalloyl residues\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCarbohydrates identified in hydrolysates after 6 hours of the biomasses used in this study by means of tested enzymatic assays. Values are the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent experiments.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eTiter (g/L)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSOW\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSOB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMTW\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMTB\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eGlucose\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eFructose\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eXylose\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eGalactose\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe wet chemical analysis (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e5\u003c/span\u003e) reveals the presence of glucose, fructose, xylose, and galactose indicating the successful hydrolysis especially of the hemicellulose component of the biomasses studied. The carbohydrates identified are in line with previous findings regarding oak-based hemicelluloses.\u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e These results are confirmed by the HSQC analyses, with the interesting exception of the xylose component in SO samples. While the wet-chemical test shows higher quantities for xylose in SO samples than MT samples, HSQC signal intensity suggests the contrary. The reason for this cannot be delineated precisely on the basis of the data available. More importantly, however, HSQC analyses were able to unveil the presence of additional carbohydrates and derivatives that the wet-chemical test could not target. \u003cem\u003eVia\u003c/em\u003e a series of characteristic cross-peaks assigned on the basis of various literature sources \u003csup\u003e\u003cspan additionalcitationids=\"CR49 CR50 CR51 CR52 CR53 CR54 CR55 CR56\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, essentially the same species were identified in all the samples, with variations in relative abundances. \u003cb\u003eSO\u003c/b\u003e-derived samples show in HSQC analyses only traces of xylose and methyl glucuronic acid. \u003cb\u003eMT\u003c/b\u003e-samples show in HSQC analyses traces in arabinose, whereas this carbohydrate is not clearly detectable in \u003cb\u003eSO\u003c/b\u003e samples. Acetylated carbohydrates have been identified in form of acetylated xylose in all samples. This is indicative of the expectable presence of acetylated xylan moieties, especially in \u003cb\u003eMT\u003c/b\u003e samples, and thus in line with one of the most famous representatives of hardwood hemicellulose features.\u003c/p\u003e\u003cp\u003eHSQC spectra indicate also the presence of alkanoates, originating eventually from waxes present in the hydrolysate. Other weak cross-peaks could be assigned to alkyl residues, eventually stemming from alkylated sugars. Most importantly, \u003cb\u003eSO\u003c/b\u003e-derived hydrolysates \u003cb\u003eSOW\u003c/b\u003e and \u003cb\u003eSOB\u003c/b\u003e contain very weak cross-peaks in the aromatic region that could potentially indicate the presence of pyrogallyl and gallate units, as such presence in tannins, with the latter especially in the typically oak-derived tannic acid. The cross-peak typical for a gallate unit is more pronounced in the \u003cb\u003eSOB\u003c/b\u003e sample, whereas here the pyrogallyl peak is hardly detectable. This is a relevant observation since propyl gallate was demonstrated to increase microbial carotenogenesis in the thraustochytrids.\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe combined NMR analyses do not indicate the presence of lipids, since typical glycerol-derived cross-peaks are absent. Neither amino acids or peptide residues were detected. Samples do contain nitrogen sources, however, as revealed by wet-chemical tests, but contents in amino acids are supposedly too low as that they would exceed the detection threshold of around 1\u0026ndash;2% (w/w) typical for NMR analyses. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows the amount of primary nitrogen source found in the hydrolysate. Nitrogen content is not increased by the action of saccharifying enzymes, with \u003cb\u003eMTB\u003c/b\u003e as an exception. The measured amount of nitrogen permits to calculate C/N ratio for the different hydrolysate, being 38 for SOW, 80 for SOB, 34 for MTW and 97 for MTB. C/N ratio is one of the factors involved in the triggering to produce lipids and carotenoids in oleaginous yeasts \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Given the presence of both carbon and nitrogen sources, the ability of \u003cem\u003eR. toruloides\u003c/em\u003e to grow in such media without any supplementation of nutrients, or elimination of possible toxic compounds was directly tested, to further improve the sustainability aspects of the present work in respect to published bioprocesses based on this microbial cell factory and residual biomasses (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.3. \u003cem\u003eR. toruloides\u003c/em\u003e production of carotenoids from woody hydrolysate\u003c/h2\u003e\u003cp\u003eOnce the presence of fermentable sugars in the hydrolysates was confirmed, the ability of \u003cem\u003eR. toruloides\u003c/em\u003e to grow in such media, withstanding possible inhibitory compounds, and to produce carotenoids was tested. As described in Section 2.7, the yeast was directly inoculated into the woody hydrolysate and sampled over time for growth, sugar consumption, and carotenoids production. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e summarises the obtained results from the growth kinetics on \u003cb\u003eSOW\u003c/b\u003e, \u003cb\u003eSOB\u003c/b\u003e, \u003cb\u003eMTW\u003c/b\u003e, and \u003cb\u003eMTB\u003c/b\u003e hydrolysates.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMTB\u003c/b\u003e is the biomass supporting the highest growth in terms of OD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), which is consistent with having the highest initial sugar content among the hydrolysates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). While \u003cem\u003eR. toruloides\u003c/em\u003e shows a similar growth profile in \u003cb\u003eMTW\u003c/b\u003e and \u003cb\u003eSOW\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and C) considering the initial difference in the provided sugars, growth in \u003cb\u003eSOB\u003c/b\u003e hydrolysate resulted to be linear until reaching a plateau (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), rather than being exponential. Since the NMR analysis of \u003cb\u003eSOB\u003c/b\u003e revealed the presence of substructures typical for tannins, above the NMR detection threshold, which is typically around 5% (w/w); it could be speculated that this tannin presence is interfering with yeast productivity. Similarly, terpenes detected in \u003cb\u003eSOB\u003c/b\u003e extractives in higher concentration and variety with respect to the other samples by GC-MS analysis (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), could have an inhibitory effect.\u003c/p\u003e\u003cp\u003eNevertheless, \u003cem\u003eR. toruloides\u003c/em\u003e was able to grow in all cases, consumed the available carbohydrates, despite not completely, and, most importantly, accumulated carotenoids. Specifically, both β-carotene and torulene were detected intracellularly, with the first being the more abundant of the two. The ratio between the two carotenoids in oleaginous yeasts may vary depending not only on strain, but also on the conditions, such as sugar availability, C/N ratio, or light exposure \u003csup\u003e\u003cspan additionalcitationids=\"CR60 CR61\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. The highest production of β-carotene occurred, in light of the above discussed inhibitors, interestingly on \u003cb\u003eSOB\u003c/b\u003e hydrolysate after 30 h of growth, reaching 362.7\u0026thinsp;\u0026plusmn;\u0026thinsp;33.9 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), followed by \u003cb\u003eMTB\u003c/b\u003e with 165.3\u0026thinsp;\u0026plusmn;\u0026thinsp;22.6 mg/L of β-carotene (after 30 h of growth) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The production of torulene was instead observed to be similar across the provided biomass around 24 and 30 h or growth, ranging from 4.5 to 2.5 mg/L. Calculation and comparison of carotenoid yields in the different conditions are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e4\u003c/span\u003e, whereas Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e permits to visualize the direct comparison between carotenoids production profiles. These data are in accordance with previous findings disclosing that a C/N ratio of 80, as in \u003cb\u003eSOB\u003c/b\u003e hydrolysate, is optimal to maximise β-carotene production over other carotenoids.\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e In addition, as already mentioned, the presence of gallates in SOB hydrolysate can be considered an additional triggering element for carotenogenesis \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eQuantitative analysis and yields calculations of β-carotene and torulene production by \u003cem\u003eR. toruloides\u003c/em\u003e from \u003cb\u003eSOW\u003c/b\u003e, \u003cb\u003eSOB\u003c/b\u003e, \u003cb\u003eMTW\u003c/b\u003e, and \u003cb\u003eMTB\u003c/b\u003e hydrolysates, after 30 h of fermentation. Values are the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent experiments.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eProduction (mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eYield on\u003c/p\u003e\u003cp\u003einitial biomass (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eg/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eYield on\u003c/p\u003e\u003cp\u003ehydrolysate sugars (g/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eYield on\u003c/p\u003e\u003cp\u003econsumed sugars (g/g)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eβ-carotene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTorulene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eβ-carotene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTorulene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eβ-carotene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTorulene (10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eβ-carotene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eTorulene (10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSOW\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e123.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e83\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0,4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSOB\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e362.7\u0026thinsp;\u0026plusmn;\u0026thinsp;33.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e241\u0026thinsp;\u0026plusmn;\u0026thinsp;23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e9,2\u0026thinsp;\u0026plusmn;\u0026thinsp;1,1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMTW\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.9\u0026thinsp;\u0026plusmn;\u0026thinsp;14.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMTB\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e163.5\u0026thinsp;\u0026plusmn;\u0026thinsp;22.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e109\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows, however, that, considering the yield on hydrolysate sugars, \u003cb\u003eSOW\u003c/b\u003e hydrolysate can be calculated to represent the best option, after 30 h of growth, for torulene production (0.047\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008% g/g), and the second best option for β-carotene production (1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20% g/g). \u003cb\u003eSOB\u003c/b\u003e hydrolysate remains the best choice considering β-carotene yields on both initial sugars in the medium and on consumed sugars (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). \u003cb\u003eMT\u003c/b\u003e-derived samples, in this calculation mode, fall behind the \u003cb\u003eSO\u003c/b\u003e-derived ones, for both β-carotene and torulene production.\u003c/p\u003e\u003cp\u003eThis is consistent with previous data on \u003cem\u003eR. toruloides\u003c/em\u003e growth on residual biomasses hydrolysates, where the maximum production of carotenoids was reached between the end of the exponential phase and the entrance of the stationary one.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e In case of \u003cb\u003eSOB\u003c/b\u003e the presence of a linear growth rather than an exponential one can be seen as indicative of the fact that the cells face hurdles, potentially due to the gallate presence (\u003cem\u003evide supra\u003c/em\u003e). Indeed, stressful compounds (such as phenolic ones), or more generally stressful growth conditions, are known to act as a trigger to production of carotenoids, while being detrimental to growth.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e Therefore, when developing bioprocesses based on residual lignocellulosic biomasses, a trade-off between these actors has to be considered to maximise both growth and carotenogenesis.\u003c/p\u003e\u003cp\u003eIn order to investigate whether also components comprised in the extractives could represent inhibitors, dedicated studies in this direction were performed. In fact, such compounds are typically present in woody biomasses (like those valorised in this study), while being limited or absent in other organic / lignocellulosic materials, such as wheat bran, tea or molasses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Effect of extractives on the production of carotenoids\u003c/h2\u003e\u003cp\u003eTo study the importance of extractives and/or impurities, \u003cb\u003eMTB\u003c/b\u003e and \u003cb\u003eSOB\u003c/b\u003e were chosen as biomasses since the numbers and respective amounts of extractives in these bark samples were higher compared to the ones found for the wood samples (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Extractives were removed as described in Section 2.13 and extracted \u003cb\u003eMTB\u003c/b\u003e and \u003cb\u003eSOB\u003c/b\u003e, \u003cem\u003ei.e.\u003c/em\u003e, \u003cb\u003eeMTB\u003c/b\u003e and \u003cb\u003eeSOB\u003c/b\u003e, respectively, were subjected to enzymatic hydrolysis and SHF following the established procedures. Data obtained from growth in terms of optical density (OD), sugar concentration over time, and carotenoids produced allowed for determining the impact of the presence or absence of extractives in the biomasses. Data are summarised in form of Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows that the elimination of extractives did not have an impact on enzymatic hydrolysis since the amount of released sugars present in the starting medium is comparable regardless of their presence. We expected the elimination of extractives might improve microbial performance and, in turn, the production of carotenoids. Indeed, \u003cb\u003eeSOB\u003c/b\u003e hydrolysate permits for a higher OD compared to \u003cb\u003eSOB\u003c/b\u003e, despite a comparable sugar consumption, suggesting that the extractives are responsible of impairing yeast growth. On the other hand, no significant difference can be observed between growth on \u003cb\u003eMTB\u003c/b\u003e and \u003cb\u003eeMTB\u003c/b\u003e. Surprisingly and independently from growth recovering, the absence of extractives in \u003cb\u003eeSOB\u003c/b\u003e and \u003cb\u003eeMTB\u003c/b\u003e resulted in a reduced accumulation of carotenoids (Fig.\u0026nbsp;6CD), compared to \u003cb\u003eSOB\u003c/b\u003e and \u003cb\u003eMTB\u003c/b\u003e derived carotenoids, implying that the presence of extractives, while being inhibitors, acts as a trigger for carotenogenesis. In fact, the production of carotenoids on eMTB and eSOB hydrolysates is comparable to previous studies whose starting feedstock for \u003cem\u003eR. toruloides\u003c/em\u003e do not typically contain significant amount of such extractives (\u003cem\u003ee.g.\u003c/em\u003e wheat bran, tea, molasses) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Indeed, some of the molecules identified in either SOB or MTB extractives (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) are associated with antimicrobial activity, like in the case of lupeol \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, β-amyrin \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e and benzaldehyde \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e possibly causing a stressful environment leading to carotenogenesis. These observations provide novelty in terms of understanding that extractive components of lignocellulosic biomasses can act as promoting elements for the production of the desired molecule, especially when it is triggered by stressful conditions (like carotenogenesis). These findings are in line with previous literature, which suggest the role of different types of stress in the production of carotenoids by \u003cem\u003eR. toruloides.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Furthermore, in terms of biorefinery development, detoxification of the initial biomass is not needed in this case, making the process simpler.\u003c/p\u003e\u003cp\u003eThese data also suggest that growth and production of carotenoids are not strictly correlated, depending on the environmental conditions. We can thus correlate microbial nutritional needs and biomass composition to the production of the desired compounds, which is definitely a relevant issue in the field of bioprocesses and biorefineries. Further investigations, beyond the scope of this work, might involve genomic and transcriptomic analysis of carotenogenesis related genes in \u003cem\u003eR. toruloides\u003c/em\u003e, to better understand the effect of extractives (as a cohort or as single compounds) on cellular behaviour and regulation.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study demonstrates the potential of leveraging diverse urban lignocellulosic residues as feedstock for microbial-based biorefineries. The findings here disclosed confirm that the enzymatic hydrolysis effectively releases fermentable sugars from previously underutilized sessile oak and mulberry tree biomasses. \u003cem\u003eR. toruloides\u003c/em\u003e exhibited robust performance, growing on these hydrolysates independently from their different sources, and efficiently accumulating carotenoids, including β-carotene and torulene. The observation that natural inhibitors present in the hydrolysates stimulate carotenoid biosynthesis underscores a critical element: challenging biomass components can, in fact, enhance certain desired product formation, optimizing the bioprocess without requiring intensive pre-treatment for inhibitor removal. Interestingly, when extractive components were removed from the biomass, carotenoids production was reduced, being more comparable with previous results from other works using other biomasses.\u003c/p\u003e\u003cp\u003eThis research contributes to the advancement of a circular bioeconomy by transforming local, readily available waste streams into valuable bio-products. It provides a robust framework for valorising biodiversity and minimizing environmental impact associated with waste disposal. Specifically, this biorefinery approach demonstrates the valorisation of regional woody biomass for the production of high-value pigments and food additives, contributing to a broader understanding of biodiversity valorisation. Future work will focus on further optimizing the bioprocess, and deepening the understanding of inhibitor-carotenogenesis interactions. Furthermore, considering the intrinsic differences between hardwood and softwood (\u003cem\u003ee.g.\u003c/em\u003e, density), which can affect enzymatic hydrolysis efficiency, investigating the effect of different parts of the same plant on the process could be a valuable area to explore. Moreover, to truly \"close the loop\" in this biorefinery concept, investigations into the valorisation of the remaining lignin-rich residues are paramount.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eS.B. declares to be part of the editorial board of Biotechnology for Biofuels and Bioproducts.The authors declare they do not have any further competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthics, Consent to Participate, and Consent to Publish\u003c/strong\u003e\u003cp\u003enot applicable.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe authors acknowledge funding under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4 - Call for tender No. 3138 of 16 December 2021, rectified by Decree n.3175 of 18 December 2021 of Italian Ministry of University and Research funded by the European Union \u0026ndash; NextGenerationEU. \u003cb\u003eAward Number\u003c/b\u003e: Project code CN_00000033, Concession Decree No. 1034 of 17 June 2022 adopted by the Italian Ministry of University and Research, CUP H43C22000530001, Project title \u0026ldquo;National Biodiversity Future Center - NBFC\u0026rdquo;.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.B., F.S., G.M.B., M.D., V.T. were involved in Investigation and Data curation.S.B., F.S., V.T., H.L., P.B. were involved in Conceptualization.S.B. was involved in Writing-Original draft preparation.S.B., F.S., V.T., D.P., M.O., H.L., P.B. were involved in Writing-Reviewing and Editing.D.P., M.O., H.L., P.B. were involved in Funding acquisition and Supervision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBertacchi, S., Jayaprakash, P., Morrissey, J. 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Antibacterial Mode of Action of β-Amyrin Promotes Apoptosis-Like Death in Escherichia coli by Producing Reactive Oxygen Species. \u003cem\u003eJ Microbiol Biotechnol\u003c/em\u003e 32, 1547 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUllah, I. \u003cem\u003eet al.\u003c/em\u003e Benzaldehyde as an insecticidal, antimicrobial, and antioxidant compound produced by Photorhabdus temperata M1021. \u003cem\u003eJournal of Microbiology\u003c/em\u003e 53, 127\u0026ndash;133 (2015).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rhodotorula toruloides, Carotenoids, Biodiversity valorisation, Biorefinery, Lignocellulosic biomass, Enzymatic hydrolysis","lastPublishedDoi":"10.21203/rs.3.rs-8187172/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8187172/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis work explores the potential of underutilized urban pruning residues from hardwood as feedstocks for bioprocesses based on the carotenogenic yeast \u003cem\u003eRhodotorula toruloides\u003c/em\u003e, investigating the correlation between biomass composition and carotenoids production. Enzymatic hydrolysates from woods and barks of sessile oak and mulberry tree were used as substrates for microbial fermentation, obtaining superior titers and productivity of β-carotene and torulene when compared to previous published processes. Mulberry tree bark hydrolysate yielded the highest total sugars (16.4 g/L), but sessile oak bark hydrolysate showed the highest β-carotene production (362.7 mg/L) after 30 hours of fermentation. Woody biomasses are known to contain significant amounts of extractive inhibitory compounds. Surprisingly, when we removed them in order to promote growth and production, we observed a significant drop in carotenoids titers, which resulted in line with published productions from biomasses lacking extractive components. These data suggest that stressful compounds present in the extractive fractions are crucial for promoting high production and productivity, when compared with the use of biomasses lacking such components. This research therefore highlights the potential of underexplored urban woody residues, thanks to the presence of triggering components, as an advantageous feedstock for microbial carotenoids production.\u003c/p\u003e","manuscriptTitle":"Rhodotorula toruloides for carotenoid production using waste hardwood biomass","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-10 10:18:44","doi":"10.21203/rs.3.rs-8187172/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-01T06:20:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-28T23:45:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-22T05:37:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279310271997964469937002005594095507997","date":"2025-12-09T09:43:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230065373305105221508402906470522256157","date":"2025-12-06T13:56:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285117622094050063445516183748954398864","date":"2025-12-05T16:24:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44425745599550323750698426177210101092","date":"2025-12-05T11:13:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-05T11:07:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-29T07:16:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-29T07:16:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology for Biofuels and Bioproducts","date":"2025-11-23T17:59:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7b315e36-dcfa-4781-9f20-7cd019d6fd50","owner":[],"postedDate":"December 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:29:46+00:00","versionOfRecord":{"articleIdentity":"rs-8187172","link":"https://doi.org/10.1186/s13068-026-02749-3","journal":{"identity":"biotechnology-for-biofuels-and-bioproducts","isVorOnly":false,"title":"Biotechnology for Biofuels and Bioproducts"},"publishedOn":"2026-03-24 16:09:16","publishedOnDateReadable":"March 24th, 2026"},"versionCreatedAt":"2025-12-10 10:18:44","video":"","vorDoi":"10.1186/s13068-026-02749-3","vorDoiUrl":"https://doi.org/10.1186/s13068-026-02749-3","workflowStages":[]},"version":"v1","identity":"rs-8187172","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8187172","identity":"rs-8187172","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}